Anchoring and uniting the An. funestus assembly for improved vector analysis. Anopheles funestus is one of the three most important and widespread vectors of human malaria in tropical Africa, but unlike An. gambiae with which it broadly co-occurs, it is a relatively neglected species. It shares with An. gambiae not only a broad sub-Saharan distribution and major vector status, but also abundant chromosomal inversion polymorphism and apparently shallow population structure across much of Africa. However, there are behavioral and genetic heterogeneities relevant to malaria transmission that remain poorly understood. In the savannas of West Africa, where application of residual insecticides in the 1960's was not as successful against An. funestus as elsewhere in Africa, there is strong cytogenetic evidence for cryptic, temporally stable assortatively mating populations co-occurring in the same villages. In apparent analogy to the chromosomal forms of An. gambiae, the chromosomally recognized forms of An. funestus, named Kiribina and Folonzo cytotypes, seem to differ in larval ecology. Importantly, they also differ in adult behaviors affecting vectorial capacity, most notably indoor/outdoor resting behavior. At present, there exist no rapid molecular identification assays to facilitate more in-depth field studies of their behavio and genetics; the cytotypes can only be distinguished by laborious chromosomal karyotyping. Our long-term goal is to understand the underlying genomic determinants of epidemiologically important phenotypic and behavioral traits in An. funestus and its cytotypes. The newly sequenced An. funestus genome begins to make possible this goal, but its fragmented state, unanchored to chromosomes, poses a barrier that hinders the identification of causal loci affecting traits of interest. The central goal of this R21 is to upgrade the draft An. funestus reference to a chromosome-based assembly in which the unanchored scaffolds are united, ordered and oriented on chromosome arms, enabling a preliminary assessment of genomic divergence between the cytotypes. Toward this end, we propose three specific Aims: 1. Integrate the assembly with the cytogenetic map by physical mapping. 2. Unite, order and orient unanchored scaffolds using single molecule sequencing. 3. Assess divergence between An. funestus cytotypes by genome scans. The innovative strategy of integrating a powerful new sequencing technology with traditional physical mapping to chromosomes will transform the draft reference into a chromosome-based assembly, and provide the means to uncover in An. funestus and its cytotypes the genetic basis of traits that affect disease transmission.